Multi-chambered cell culture device to model organ  microphysiology

ABSTRACT

A cell culture device can include: a top wall; a bottom wall; one or more perimeter walls coupled with and extending between the top wall and bottom wall; and at least 3 distinct chambers between the top wall, bottom wall. The one or more perimeter walls can include: an internal chamber defined by at least one porous internal wall and having an internal chamber inlet and an internal chamber outlet; one or more boundary layer chambers having at least an inner boundary layer chamber defined by the at least one porous internal wall and at least one porous inner boundary layer wall, the at least one porous internal wall having a plurality of pores fluidically coupling the central internal chamber to the one or more boundary layer chamber; and an outer chamber defined by an outer porous boundary layer wall of the at least one porous boundary layer walls and the one or more perimeter walls and having an outer chamber inlet and an outer chamber outlet, the outer porous boundary layer wall having a plurality of pores that fluidically couple the outer chamber with the one or more boundary layer chambers. In one aspect, wherein the at least three distinct chambers are nonlinear and/or idealized.

CROSS-REFERENCE

This patent application claims priority to U.S. Provisional PatentApplication 61/730,357 filed Nov. 27, 2012, which provisionalapplication is incorporated herein by specific reference in itsentirety.

BACKGROUND

Current in vitro platforms are poor predictors of in vivo safety,efficacy and pharmacokinetics of therapeutic agents or therapeuticdelivery systems having therapeutic agents owing to significantdifference in the test conditions compared to physiological conditions.Traditional in vitro models routinely utilize 2D monolayers of culturedcells under static conditions for studying drug delivery and toxicity.These simplistic representations often result in waste metabolitebuildup in the platform, which can provide misleading information on thephysiological condition. In order to overcome this limitation, perfusedcell culture systems or bioreactors were developed to continuouslyreplenish the culture medium. However, the use of continuously fedsystem leads to cost prohibitive reagent volume requirements.Microfluidic bioreactors were developed to address this challenge, andoffer several key advantages over conventional macro-physiologicalsystems (e.g., hollow fiber or membrane-based technologies). Forinstance, microfluidic systems offer facile compatibility withco-culture conditions, in particular multi-cellular architectures,real-time optical monitoring, and a more accurate representation ofcell-cell interactions. However, available biomicroreactors fail tocapture key in vivo physiological features such as morphological size,physiological blood flow and cellular (biological) architecture of thespecific organs being investigated. Therefore, there remains a need forimproved cell culture devices in order to enable improved modeling oforgan and/or physiological response to therapeutic agents or therapeuticdelivery systems having therapeutic agents.

FIGURES

The foregoing and following information as well as other features ofthis disclosure will become more fully apparent from the followingdescription and appended claims, taken in conjunction with theaccompanying drawings. Understanding that these drawings depict onlyseveral embodiments in accordance with the disclosure and are,therefore, not to be considered limiting of its scope, the disclosurewill be described with additional specificity and detail through use ofthe accompanying drawings, in which:

FIG. 1A illustrates an embodiment of a multi-chamber cell culture devicein an idealized configuration.

FIG. 1B illustrates lateral cross-sectional views of embodiments ofmulti-chamber cell culture devices having idealized configurations.

FIG. 2 illustrates an embodiment of a multi-chamber cell culture devicein a synthetic microvascular network (SMN) configuration.

FIG. 3 illustrates an embodiment of a multi-chamber cell culture devicehaving inlets and outlets having idealized configurations and themulti-chambers in a synthetic microvascular network (SMN) configuration.

FIG. 4 illustrates an embodiment of a multi-chamber cell culture devicehaving inlets and outlets having synthetic microvascular network (SMN)configurations and the multi-chambers having an idealized configuration.

FIG. 5 illustrates an embodiment of a multi-chamber cell culture devicewith a pass-through passageway in an idealized configuration.

FIG. 6 illustrates an embodiment of a multi-chamber cell culture devicewith a non-planar inlets and outlets in an idealized configuration.

FIG. 7 illustrates an embodiment of a multi-chamber cell culture devicewith a central chamber with a planar inlet and non-planar outlet withlateral chambers having circumferential pathways with inlets and outletson a common side in an idealized configuration.

FIG. 8 illustrates an embodiment of a multi-chamber cell culture devicewith central chamber with internal pillars or posts and barrier pillarsor posts forming porous walls between the chambers in an idealizedconfiguration.

FIGS. 9A-9C illustrate different embodiments of barrier pillars or postsforming porous walls.

FIG. 10 illustrates an embodiment of SMN network having SMN fluidpathways and SMN multi-chambered cell culture constructs.

FIG. 11 illustrates an embodiment of a network showing parallel and/orseries multi-chambered cell culture constructs.

FIG. 12 illustrates an embodiment of parallel and series multi-chamberedcell culture constructs having bifurcated outlets and joining inlets.

All aspects of the embodiments described in the figures can be used inconjunction with other embodiments in other figures. For example, postscan be used in a barrier layer or barrier conduit without being used inthe central or interior chamber. Also, the figures are not to scale ordimension. For example, the distances between chambers may be differentwith respect to other distances between other chambers. While someaspects are shown to be symmetrical, those aspects may be asymmetrical.

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying drawings, which form a part hereof. In the drawings,similar symbols typically identify similar components, unless contextdictates otherwise. The illustrative embodiments described in thedetailed description, drawings, and claims are not meant to be limiting.Other embodiments may be utilized, and other changes may be made,without departing from the spirit or scope of the subject matterpresented herein. It will be readily understood that the aspects of thepresent disclosure, as generally described herein, and illustrated inthe figures, can be arranged, substituted, combined, separated, anddesigned in a wide variety of different configurations, all of which areexplicitly contemplated herein.

Generally, the present invention relates to a multi-chamber cell culturedevice that can provide improved organ models as well as methods ofmaking and using the same. The multi-chamber cell culture device caninclude an outer chamber that can be used as an outer conduit and aninternal chamber that can be used as an internal organ tissue spaceseparated by one or more barrier chambers (e.g., barrier layerchambers). As such, the multi-chamber cell culture device can be aconstruct with an outer chamber and a central chamber separated by oneor more barrier chambers there between. The multi-chamber cell culturedevice can include one or more of the outer chamber, one or more barrierchambers, and central being a cell culture chamber. The multi-chambercell culture device can include one or more of the outer chamber, one ormore barrier chambers, and central being devoid of cell cultures. Themulti-chamber cell culture device can include an outer conduit (e.g.,outer chamber) and an internal organ tissue space (e.g., centralchamber) separated by one or more barrier layers (e.g., barrierchambers). In one aspect, an internal organ tissue space (e.g., centralchamber) may be offset or off-center or asymmetrical with respect to theouter chamber and barrier chambers, and thereby may be referenced as aninternal chamber that is surrounded by the barrier chambers that aresurrounded by the outer chamber. This provides the chambers in an onionlayer arrangement.

The conduit, organ tissue space, and one or more organ barrier layerscan be distinct chambers that are partitioned from each other withporous walls. The porous walls can have true pores or have gaps betweenwall sections or gaps between barrier pillars or posts that function aspores so that fluid and nutrients and test analytes can pass between thedistinct chambers. In one option, the pores can be gaps that are largeenough for cancer cells to pass therethrough, such as for cancermetastasis modeling or for cell migration. The porous walls can beconfigured to keep the chambers distinct from each other while the poresin the porous walls can allow for nutrients to move therebetween. Thisconfiguration can provide for modeling of an organ. Generally, any ofthe chambers, such as the outer chamber, inner chamber, boundary layerchambers, inner organ tissue space chamber, one or more organ barrierlayers, or other distinct region in the multi-chamber construct can bedistinct chambers that are partitioned from each other with porous wallsand used for tissue culture spaces. For example, the outer chamberand/or any barrier chamber and/or any combination thereof with orwithout the internal chamber can be used as a tissue culture chamber.Oppositely, the outer chamber and/or any barrier chamber and/or anycombination thereof with or without the central chamber can be usedwithout having any cell culture therein.

The incorporated references describe idealized microvascular networks(IMN) and synthetic microvascular networks (SMN), which can be includedin the inlets, outlets or chambers therebetween. That is, an IMN caninclude one or more multi-chambered cell culture constructs in an IMNconfiguration; or a SMN can include one or more multi-chambered cellculture constructs in an SMN configuration; or a hybrid IMN/SMN caninclude fluid pathways that include features of IMN and/or SMN and oneor more multi-chambered cell culture constructs with the IMN or SMNconfiguration. Accordingly, the multi-chamber cell culture device can beconfigured with distinct chambers that are modeled by IMN and/or SMN.Some configurations can include only IMN chambers, some may include onlySMN chambers, and some can include a combination of both IMN and SMNchambers. The multi-chamber cell culture device can be configured forany organ with an appropriate outer conduit simulating an organ, one ormore barrier layer conduits simulating a barrier membrane of an organ,and an organ tissue space configured as the tissue that provides theorgan function. For example, the multi-chambered cell culture device cansimulate the liver, kidney, heart, lung, brain, stomach, intestine,blood brain barrier, vascular networks, or others. As such, the distinctchambers can have unique cell cultures that are indicative of thedifferent cell types or tissue types of the different layers and centralregion of an organ, where the cell culture in the outer conduit chambercan be different from the organ barrier layer chambers, which can bedifferent from the internal organ tissue chamber. A single embodiment ofthe multi-chambered cell culture device can be configured to bedifferent organs by the different cells or cell combinations that arepresent in the distinct cell cultures of the distinct chambers. That is,different types of cells and cell combinations can distinguish a devicesimulating the heart from a device simulating the liver, where withoutthe cells the devices can appear similar or identical.

The multi-chambered cell culture device can include a fluid inlet andfluid outlet for each of the distinct chambers. The fluid inlets andoutlets can be adjacent or distributed about the device, or random onthe device. The fluid outlet of one device can be fluidly coupled to theinlet of another device so that multiple simulated organs can be linked(see FIG. 10). For example, a metabolic pathway or organ series can bemimicked by linking multiple devices through their inlets and outlets.For example, a series of simulated organ devices can be: lung; liver;heart; and kidney. The linked devices may be in series and/or inparallel (see FIG. 11), and may be linear or may include branches (seeFIGS. 11 and 12). For example, a liver device may be fluidly coupled toa downstream brain device and a kidney device. As such, the fluid inletsand outlets can be bifurcated. Such bifurcations can be idealized (e.g.,IMN) or synthetic (e.g., SMN). The multi-chambered cell culture devicecan include one or more IMN or SMN networks coupled to one or more ofthe inlets or outlets of one or more of the individual chambers of themulti-chambers.

The multi-chambered cell culture device can be configured to be retainedin any common cell culture incubator or other common laboratoryequipment used for growing, propagating, and analyzing cell cultures.The inlets and outlets can be configured to be coupled to tubing, cellculture pumps, syringe pumps, or other cell culture equipment or pumpsthat can move fluid through the fluid inlets and outlets as well asthrough the distinct chambers. Unique pumps can be coupled to thedifferent chambers.

FIG. 1A shows an embodiment of a multi-chambered cell culture device 100in accordance with the principles of the present invention. Themulti-chambered cell culture device 100 is shown to include an internalchamber 110 (e.g., internal organ tissue chamber), an inner boundarylayer chamber 120, an outer boundary layer chamber 130, and an outerconduit layer chamber 140. However, only one boundary layer chamber ormore than two additional boundary layer chambers can be located betweenthe internal chamber 110 and outer conduit layer chamber 140. That is,the multi-chambered cell culture device 100 can include one or moreinternal chambers 110 (or central chambers), one or more inner boundarylayer chambers 120, one or more outer boundary layer chambers 130, andone or more outer conduit layer chamber 140. As such, any of thechambers can be partitioned into a plurality of chambers with porouswalls. It should be noted that the internal chamber 110 is designated asa tissue chamber due to certain use embodiments; however, such internalchamber 110 may not be used as a tissue chamber or cell culture chamber,and may be used as an internal or central chamber. The internal chambermay also be used for 2D cell cultures or monolayers, which can beapplied to any of the chambers. The internal chamber 110 can include afluid inlet 112 and a fluid outlet 114. The inner boundary layer chamber120 can include at least one fluid inlet 122 a and 122 b, which can befluidly coupled or fluidly separate, and include at least one fluidoutlet 124 a and 124 b, which can be fluidly coupled or fluidlyseparate. The outer boundary layer chamber 130 can include at least onefluid inlet 132 a and 132 b, which can be fluidly coupled or fluidlyseparate, and include at least one fluid outlet 134 a and 134 b, whichcan be fluidly coupled or fluidly separate. The outer conduit layerchamber 140 can include at least one fluid inlet 142 a and 142 b, whichcan be fluidly coupled or fluidly separate, and include at least onefluid outlet 144 a and 144 b, which can be fluidly coupled or fluidlyseparate. The internal chamber 110 can be defined by a porous tissuechamber wall 116, the inner boundary layer chamber 120 can be defined bythe porous tissue chamber wall 116 and a porous boundary layer wall 126,the outer boundary layer chamber 130 can be defined by the porousboundary layer wall 126 and a porous outer conduit wall 136, and theouter conduit layer chamber 140 is defined by the porous outer conduitwall 136 and an external wall 102 that is not porous. As also shown, theexternal wall 102 can be a side wall between a top wall 104 and a bottomwall 106, which may be coupled or integrated. For example, the bottomwall 106 can be integrated with the external wall 102 and porous walls,and the top wall 104 can be coupled to the external wall and porouswalls. The top wall 104 can be a lid. The right and left sides cancooperate to form the boundary layer chamber and outer conduit chamber,or they may be distinct right and left side chambers that are configuredas boundary layer chambers or outer conduit chambers. While notspecifically shown, the inlets and outlets may each individually includeinlet valves and outlet valves, which can be selectively opened to allowfluid flow or pressure and closed for incubation.

While the inlets 122 a and 122 b for the inner boundary layer chamber120 are shown to be separate, they may be fluidically coupled. Also, theoutlets 122 a and 122 b may also be separate or fluidically coupled. Theother inlets and outlets for the discrete chambers may also be separateor fluidically coupled. Such fluid coupling can be by tubing or otherfluid conduits coupled to both inlets or both outlets, or the device canbe structured in three-dimensions with a fluid pathway passing over orunder a different fluid pathway. Also, while the discrete chambers areshown to have left sides that are separate from right sides, the lateralchambers may indeed be separate or they may be fluidically coupled bypassing the individual chambers over or under other chambers. A myriadof design choices are available under the teachings provided herein.

FIG. 1B shows a linear multi-chamber embodiment 100 a where the inletsand/or chambers and/or outlets are separate and aligned in a plane.Also, a squared concentric multi-chamber embodiment 100 b where theinlets and/or chambers and/or outlets are squared concentric, and isshown with the internal chamber outlet 114 being squared concentric withthe other outlets 124, 134, and 144. Also, the inlets and/or chambersand/or outlets can be round concentric with round cross-sectionalprofiles as shown in round concentric multi-chamber embodiment 100 c, orconcentric with polygon cross-sectional profiles. Additionally, theinlets and/or chambers and/or outlets are can be semi-concentric as insemi-concentric multi-chamber embodiment 100 d with a common top wall104. Also, the outlet configurations of FIG. 1B can be applied to theinlets as well as to the body of the device that has the distinctconduits. That is, any of the inlets, outlets, or the distinct conduitscould be arranged as illustrated in FIG. 1B, or any combination of theillustrations, e.g., round semi concentric.

While FIG. 1A shows a generally idealized configuration that may includefeatures of an IMN network, FIG. 2 shows the multi-chambered cellculture device 200 can include irregular or simulated inlets, chambers,and outlets, such as in an SMN. Here, the internal chamber 210, innerbarrier layer chamber, 220, outer barrier layer chamber 230, and outerconduit layer 240 can all be irregular or modeled after an SMN network.FIG. 3 shows that device 300 can include IMN inlets and outlets with theinternal chamber 310, inner barrier layer chamber, 320, outer barrierlayer chamber 330, and outer conduit layer 340 being irregular orSMN-like. FIG. 4 shows the device 400 can include SMN inlets and outletswith the internal chamber 410, inner barrier layer chamber, 420, outerbarrier layer chamber 430, and outer conduit layer 440 being idealizedor IMN-like. However, the IMN and/or SMN configurations and shapingshown in FIGS. 1A and 2-4 can be combined or used with other devices asdescribed herein. For example, some of the distinct chambers may be IMNand some may be SMN, which also may be applied to some of the inlets andoutlets.

FIG. 5 shows a multi-chambered cell culture device 500 that has aninternal conduit 550 that is within the internal chamber 510. The innerbarrier layer chamber, 520, outer barrier layer chamber 530, and outerconduit layer 540 are configured as described herein. Here, the internalconduit 550 can be defined by a porous wall 556 such that fluid and/ornutrients can pass between the internal conduit 550 and the internalchamber 510.

FIG. 6 shows another multi-chambered cell culture device 600 configuredsimilar to other embodiments shown herein with the internal chamber 610having an inlet 612 and outlet 614 that are configured similar to fluidpathways in the substrate (e.g., bottom wall). The outer chamber 660also has one or more inlets 662 a and 662 b and one or more outlets 664a and 664 b that are configured similar to fluid pathways in thesubstrate (e.g., bottom wall). However, the boundary layer chambers 620,630, 640, and 650 have port inlets (e.g., 622 a and 622 b, 632 a and 632b, 642 a and 642 b, and 652 a and 652 b) that may or may not includeport valves, and port outlets (e.g., 624 a and 624 b, 634 a and 634 b,644 a and 644 b, and 654 a and 654 b) that may or may not include portvalves. Tubing or other fluid pathways may be coupled to the inletsand/or outlets, which may further be coupled to pumps, fluid reservoirs,media reservoirs, or other cell culture components. Also, any of theinlets can be fluidly coupled or fluidly separate upstream from theinlet, and any of the outlets can be fluidly coupled or fluidly separatedownstream from the outlets as described herein. FIG. 7 shows anotherembodiment of a multi-chambered cell culture device 700, which includesan internal chamber 710 having a fluid pathway inlet 712 and a portoutlet 714, which may include a tubing with or without a valve. Thebarrier layer chambers (e.g., 720, 730, and 740) can wrap around theinternal chamber 710 and include inlets (e.g., 722, 732, and 742) on oneside of the fluid pathway inlet 712 and outlets (e.g., 724, 734, and744) on the other side of the fluid pathway inlet 712. This can be acircumferential pathway configuration with inlets and outlets on acommon side or end. The outer chamber 750 is also circumferentiallypositioned and includes similarly arranged inlet 752 and outlet 754.Here, the outer chamber 750 is in fluid communication with the barrierlayer chamber 740 from the inlets to the outlets, and the barrier layerchamber 720 is in fluid communication with the internal chamber 710 fromthe inlets to the outlets. The internal walls are porous, which allowsthe analytes to pass between the distinct chambers from the inletthrough to the outlet as well as the cells in one chamber to communicatewith cells in the adjacent chamber. The porous walls can be configuredto provide a diffusion barrier between adjacent chambers. This canprovide a diffusion barrier between the vascular space (e.g., outerconduit chamber) and tissue space (e.g., internal chamber).

FIG. 8 shows another embodiment of a multi-chambered structure 800 inaccordance with the principles of the present invention. Themulti-chambered cell culture device 800 is shown to include an internalchamber 810, an inner boundary layer chamber 820, an outer boundarylayer chamber 830, and an outer conduit layer 840. However, only oneboundary layer chamber or more than two additional boundary layerchambers can be located between the internal chamber 810 and outerconduit layer 840. The internal chamber 810 can include a fluid inlet812 and a fluid outlet 814. The inner boundary layer chamber 820 caninclude at least one fluid inlet and at least one fluid outlet asdescribed herein. The outer boundary layer chamber 830 can include atleast one fluid inlet and at least one fluid outlet as described herein.The outer conduit layer 840 can include at least one fluid inlet and atleast one fluid outlet as described herein. The internal chamber 810 canbe defined by a porous tissue chamber wall 816, the inner boundary layerchamber 820 can be defined by the porous tissue chamber wall 816 and aporous boundary layer wall 826, the outer boundary layer chamber 830 canbe defined by the porous boundary layer wall 826 and a porous outerconduit wall 836, and the outer conduit layer 840 is defined by theporous outer conduit wall 836 and an external wall 802 that is notporous. Here, the porous walls 816, 826, 836 can include a plurality ofposts 860 that form the walls with the gaps between the posts 860. Theporous walls 816, 826, 836 have one or more posts 860 laterally orradially oriented to form the walls. FIGS. 9A-9C the porous walls caninclude any number of laterally or radially oriented posts 860 betweenchambers, where 4 posts, 6 posts, and 8 post embodiments are shown, butany number of posts 860 can be used as the porous walls, including aline of single posts 860 forming the porous walls 816, 826, 836.

In one embodiment, the porous walls 816, 826, 836 can be configured asconduits or chambers, and may include inlets 816 a, 826 a, 836 a, and/oroutlets 816 b, 826 b, 836 b. In one embodiment, the porous walls 816,826, 836 are not conduits or chambers, and are configured as barrierwalls. Here, the porous walls 816, 826, 836 may be devoid of inlets 816a, 826 a, 836 a, and/or outlets 816 b, 826 b, 836 b. As shown for porouswall 816, an inlet barrier wall 818 a and/or outlet barrier wall 818 bcan be included.

It should be recognized that the porous walls having the one or morelateral or radial posts, post array, post line, or any other orientationor distribution of posts 860 can be applied to any of the barrier wallsbetween any of the chambers/conduits of any of the other embodiments orfigures herein. The posts can have an even spacing therebetween orasymmetrical spacing. The posts can be uniform in size or have sizedistributions along a pathway or chamber or through the wall from onechamber to anther chamber.

In one embodiment, any of the chambers/conduits can include structureposts 850 that can be used to provide structure between top walls andbottom walls. The structure posts 850 can be coupled to a bottom wall,and may be coupled to a top wall when integrated with the side walls.Also, the top wall as a lid can rest on the structure posts 850. Thestructure posts can be used for cell culture, and can result in a highercell density for organ simulations. FIG. 8 shows the central chamber 810as having the posts 850, but it can be devoid of posts. Any of theboundary chambers 820, 830 can include the posts 850 or be devoid ofposts. The outer chamber 840 can include the posts 850 or be devoid ofposts.

FIG. 10 illustrates a SMN 10 having one of more fluid inlets In 9 andone or more fluid outlets Out 9 with one or more multi-chamberconstructs 1, 2, 3, 4, each having a central chamber 8 a, 8 b, 8 c, 8 d(e.g., while four multi-chamber constructs are shown, any integer can beused). The multi-chamber constructs 1, 2, 3, 4 can be configured withinlets and outlets in accordance with any of the embodiments or figuresdescribed herein. Also, while shown to be SMN, the configuration can bean IMN. The SMN can be configured with any number of fluid pathways 7linking the multi-chamber constructs, which can be in any manner, andwhich SMN can be designed via simulation of real biological orartificial fluid pathways.

As shown, multi-chamber construct 1 can include a central chamber 8 asurrounded by an outer conduit layer 1 a with barrier layer chambers 1b, 1 c therebetween. The outer conduit layer 1 a can be fluidly coupledwith an inlet In 9 and an outlet Out 9. Also, the outer conduit layer 1a can include an inlet In 1 a and an outlet Out 1 a. The barrier layerchambers 1 b, 1 c, can include inlets In 1 b, In 1 c and outlets Out 1b, Out 1 c, respectively. While not shown, the central chamber 8 a caninclude inlets or outlets, or it can receive content from the barrierlayer 1 c.

As shown, multi-chamber construct 2 can include a central chamber 8 bsurrounded by an outer conduit layer 2 a with barrier layer chambers 2b, 2 c therebetween. The outer conduit layer 2 a can be fluidly coupledwith an inlet In 9 and an outlet Out 9. Also, the outer conduit layer 2a can include an inlet In 2 a and an outlet Out 2 a. The barrier layerchambers 2 b, 2 c, can include inlets In 2 b, In 2 c and outlets Out 2b, Out 2 c, respectively. While not shown, the central chamber 8 b caninclude inlets or outlets, or it can receive content from the barrierlayer 2 c.

As shown, multi-chamber construct 3 can include a central chamber 8 csurrounded by an outer conduit layer 3 a with barrier layer chambers 3b, 3 c therebetween. The outer conduit layer 3 a can be fluidly coupledwith an inlet In 9 and an outlet Out 9. Also, the outer conduit layer 3a can include an inlet In 3 a and an outlet Out 3 a. The barrier layerchambers 3 b, 3 c, can include inlets In 3 b, In 3 c and outlets Out 3b, Out 3 c, respectively. While not shown, the central chamber 8 a caninclude inlets or outlets, or it can receive content from the barrierlayer 3 c.

As shown, multi-chamber construct 4 can include a central chamber 8 dsurrounded by an outer conduit layer 4 a with barrier layer chambers 4b, 4 c therebetween. The outer conduit layer 4 a can be fluidly coupledwith an inlet In 9 and an outlet Out 9. Also, the outer conduit layer 4a can include an inlet In 4 a and an outlet Out 4 a. The barrier layerchambers 4 b, 4 c, can include inlets In 4 b, In 4 c and outlets Out 4b, Out 4 c, respectively. While not shown, the central chamber 8 d caninclude inlets or outlets, or it can receive content from the barrierlayer 4 c.

FIG. 11 shows a network 1100 having multi-chamber constructs 1110 inparallel and series. Any of the connecting fluid pathways 1104 areoptional. While a single inlet 1112 is shown for parallel analysis, eachchain 1102 can include its own inlet 1114, and each multi-chamberconstruct 1110 can include its own inlet 1116 and central port 1118. Thecentral port 1118 can be an inlet or outlet. The network 1110 can haveindividual outlets 1120 for each chain 1102; however, the outlets 1120may be fluidly coupled into a single outlet in some instances. Any ofthe inlets 1116 or central ports 1118 can be optional. Also, the inlet1112 and outlets 1120 may be inverted. This network may be IMN or SMN.

FIG. 12 includes a network 1200 having multi-chamber constructs 1110 inparallel and series. The connecting fluid pathways 1104 are shown sothat the left chain includes a first multi-chamber construct 1110 withthe outlet fluidly coupled with two multi-chambers constructs 1110 thatare then each coupled to a single multi-chamber construct 1110. Theright chain 1102 shows two first multi-chamber constructs 1110 withtheir outlets fluidly coupled with a single multi-chambers construct1110 that is then coupled to a single multi-chamber construct 1110. Thisshows that any network arrangement of a plurality of multi-chamberconstructs in parallel and/or series can be constructed, where inletsand outlets can be bifurcated, joined, branched, or other configurationin accordance with the description herein and in the incorporatedreferences.

In one embodiment, the distinct chambers can have different cells orcell combinations for different cell cultures. The cell culture of eachchamber can have a distinct function. For example, the outer chamber canhave endothelial cells and simulate the outside of an organ, the barrierlayer conduits can have cells that simulate the barrier layers of anorgan, and the internal chamber can have cells that simulate thefunctionality of the organ.

The internal walls between the conduit chamber and organ tissue chambercan be porous so that fluid and/or nutrients can pass therebetween. Inone option, the pores can be a dimension that is too small for cells topass through; however, the pores can be enlarged in some embodiments sothat cells may pass therethrough such as when modeling cancer cellmigration or metastasis. In any event, various analytes, such as testanalytes and metabolic analytes can pass through the pores of the porouswalls. This can allow for a sequential encounter between an analyte ormetabolites therefrom from the conduit chamber, through the barrierlayer chambers, and then the organ tissue chamber. The dimension of thepores can vary. For example, the dimension of the pores can have adimension up to 50 microns and as small as 100 nm; however, thedimension can range from about 200 nm to about 30 microns. The largerpores can be for cancer tissue modeling and allow for metastasis orcancer cells migrating between chambers. Generally, the pores can besmaller than 20 microns or smaller than 10 microns to control cellmigration therethrough.

The pores can be symmetrical or asymmetric, ordered or random. The poresizes can increase or decrease from one end (e.g., inlet) to other end(e.g., outlet) of pathway, or have size gradients therebetween,parabolic distributions therebetween, or any other distribution thatprovides a porous pathway between chambers. The pores on one side of themulti-chambered construct can be larger or smaller than the pores on theother side of the construct. The pores between the outer chamber andbarrier chamber may be larger or smaller than the pores between thecentral chamber and barrier chamber. The pores between adjacent barrierchambers can be larger or smaller compared to the pores between theouter chamber and barrier chamber and/or central chamber and barrierchamber. In one example, the pores sizes decrease from the outer chamberto barrier chambers to central chambers. In another example, the poresizes increase from the outer chamber to barrier chambers to centralchambers.

The dimensions of the bottom wall, side walls, and/or top walls canrange from about 5 microns to about 400 microns or up to about 500microns, and possibly up to about 700 microns. The separation dimensionbetween side walls can be about 5 microns, about 10 microns, about 25microns, about 50 microns, about 100 microns, about 200 microns, about250 microns, or about 400 microns, or any dimension therebetween. In oneexample, the height of the side walls can be about 5 microns to about150 microns. The dimensions of the bottom and top walls of the chamberscan be the same or different. For example, the chambers can be smallerfrom the outside in or from the inside out. The perforated walls canhave a have a thickness that generally ranges from about 5 microns toabout 500 microns, or such as for example 1 micron, 10 microns, 20microns, 30 microns or up to 100 microns. In one example, the outsidechannel may be 100 microns wide, and then the barrier chambers may bemay be 200 microns wide. In one example, the barrier layers can bestaggered with dimensions of 25 microns, 50 microns, 75 microns, and 100microns. The number of barrier layer chambers can vary depending on theorgan to simulate as well as the dimensions of the chambers. Eachchamber can be large enough to culture enough cells to get somemeaningful data during an assay, but the chambers should not be toolarge where diffusion times are too great and inhibit obtainingmeaningful data.

The dimensions of the outer chamber and barrier chambers may be the sameor different, which may include the dimension from one wall to anotherwall thereof. The thickness of the walls between the outer chamber andbarrier chambers or between the different barrier chambers or betweenthe barrier chambers and central chamber may be the same or different.These dimensions can increase from the outside to the central chamber orthey may decrease from the outside to the central chamber, such as in anincreasing or decreasing size gradient for the chambers and/or walls.The dimensions may also be parabolic with increasing to decreasing toincreasing, or from decreasing to increasing to decreasing relativedimensions of the chamber and/or walls.

Virtual experiments using Computational Fluid Dynamics (CFD) or othercomputational modeling allow the ability to optimize the experimentalprotocols. This procedure not only saves time but also reduces reagentconsumption. CFD modeling can also be used to differentiate betweenperfusion based vs. diffusion based experiments in addition todetermining the flow rate ranges for optimal cell growth. CFD modelingcan also drive design optimization of each of the conduits and layers ofthe device ranging from distance for diffusion, pore size, number ofpores.

The porous walls can be configured to provide a diffusion barrierbetween adjacent chambers. This can provide a diffusion barrier betweenthe vascular space (e.g., outer conduit chamber) and tissue space (e.g.,internal organ tissue chamber).

The different chambers can be arranged in a side-by-side format asillustrated, but other configurations, such as stacked, sandwiched,volumetric, or concentric can be employed. In one embodiment, thedifferent chambers can be arranged similar to the layers of an onion.Generally, the outer chamber is used for introducing test analytes;however, the inner chamber can be used for introducing test analytes. Insome options, the boundary layer chambers can be used for introducingtest analytes as each boundary conduit can have unique inlets and/oroutlets. Alternatively, all of the boundary conduits can have a commoninlet and a common outlet. When an internal conduit is included, it canbe used for introducing test analytes directly to the organ tissuechamber for an organ tissue simulation. Thus, generally the inner mostconduit or outermost conduit can serve to provide the test analytes,which test analytes or metabolites thereof can pass through the boundarylayers by passing through the pores. Also, the conduits can beconfigured to selectively provide different nutrients to the differenttypes of cell cultures, where the outer conduit (endothelial cells) andinternal organ tissue chamber (tissue cells) receive different nutrientscompared to one or more boundary layers. Each boundary layer may receivedifferent nutrients, or they can all receive the same nutrients.

The internal chamber is shown to have an inlet fluid pathway that opensinto a space that is larger than the inlet fluid pathway. The internalchamber can be wider with a larger cross-sectional profile than theinlet and outlet fluid pathways. The porous internal walls that definethe internal chamber can diverge from each other from the inlet towardthe outlet until a medial point at which the porous internal wallsconverge toward each other until reaching the outlet. The divergence andconvergence can result in a spherical, oval, oblong, or polygon shape,such as a hexagon. The porous walls defining the boundary layers andouter conduit layer can have the same shaping as the porous internalwalls, and may have the same divergence and convergence between theinlets and outlets. In one embodiment, the entirety of the walls betweenthe internal chamber and outer non-porous perimeter wall can be porouswalls that are porous from the inlets to the outlets. Thecross-sectional profile or distance between the porous internal wallscan be from about 1.5 to 50 times larger than the boundary layerchambers cross-sectional profiles or distance between the porous wallsthat define the boundary layer chambers. The cross-sectional profile ordistance between the porous internal walls can be from about 1.5 to 50times larger than the outer chamber cross-sectional profiles or distancebetween the porous walls that define the outer chambers.

In one embodiment, the outer chambers and boundary chambers can bepartitioned into right and left sides as shown in FIG. 1A. In oneoption, the internal chamber can be partitioned into right and leftsides as shown in FIG. 5 when an internal conduit is included. The rightand left chamber sides can be considered together to be a completechamber. The right and left chamber sides can be fluidly coupled such asby having a common inlet and a common outlet or a flow path that fluidlycouples the right and left sides. However, the right and left chambersides may be separate chambers with separate inlets and separate outletsfrom each other. The right and left chambers sides can be used for thesame studies or test analytes, or different test analytes can beintroduced into the right and left chambers.

The cells can grow only on the bottom, or can grow to confluence on thesides and optionally the top walls. As such, the cells can grow over thepores. Preferably, the cells grow completely around the chambers to forma cellular lumen or three-dimensional tissues. Tissue culture scaffoldmaterials can be located in any or all of the chambers as desired. Thecells can grow over the pores but allow analytes or metabolites or otherfluid to pass through the pores to an adjacent chamber. So, the cellsstart growing at the bottom first, but eventually they fill up theporous side walls and the top walls and all around the chambers.

In a preferred embodiment, the distinct chambers can be side-by-side orhorizontally sandwiched. The device can include the chambers being thesame height so that the height is maintained across each chamber. Also,the device can be prepared so that the chambers are parallel to eachfrom the outside chamber to the internal chamber. The device can includean outside conduit or outside chamber on the outside of the device thatfunctions as a conduit, where the chamber has a bottom surface and twosidewalls—one is an outside perimeter side wall and one is an insideperimeter side wall that has perforations or is otherwise porous. Eachof the other chambers can be similarly configured with all internalwalls being porous. A top wall can enclose all of the chambers as shown.

Each chamber can provide a tissue space that includes tissue culturescaffolds that will help grow the cells in a 2D monolayer or in a 3Dsense to substantially filling up the entire space of the chamber. Inthe absence of the scaffolds, the cells will just cover the walls (e.g.,bottom, side and/or top). Whereas the tissue scaffolds provide a 3D cellculture in a nice, packed structure in the tissue space. The scaffoldscan be the same or different material from the walls. The scaffolds canbe integrated or coupled with the walls, or inserted into the chambers.The scaffolds can be cast in the same method when the walls of thechambers are cast.

The multi-chambered cell culture device can be manufactured inaccordance with known principles, such as in the incorporatedreferences. The devices can be made by providing a master and thenpouring a polymer over it, then bake or otherwise curing the polymer sothe polymer hardens, and then peeling the polymer from the master (e.g.,mold). There can be a negative of the device on the master, whichprovides the device when cast with the polymer. The device can then beattached to a top wall, such as polymer or glass. The stamp or mold canbe prepared to define all the features of the device. Also, the chamberscan include collagen or matrigel or other gelatinous cell culturescaffold material. In addition, natural or synthetic culture scaffoldscan be used. Also, electrospun fibers (comprising of culture matrix,proteins and other biological and artificial components) can be used forthe scaffolds. Cells can be mixed with scaffolds on cultured onscaffolds for creating a 2D or 3D culture.

In one embodiment, a plate can include a plurality of multi-chamberedcell culture wells. That is, a well plate can include a plurality ofwells configured with the multiple chambers as described herein. Thewells can be engineered with appropriate inlets and outlets as describedherein. The plate may include the inlets and outlets between the wells.For example, a well plate of a standard size that fits in a plate readerhaving 96 wells can be configured to include a multi-chambered cellculture well in one or more wells or in each of the 96 wells. Wellplates with smaller numbers of multi-chambered wells may also beprepared. The multi-chambered cell culture wells can be configured thesame or different. For example, a single well plate can be configuredwith two or more different organ models, which may be separate, orlinked in a biologically relevant series. The multi-chambered device canbe configured as a well, or configured as an insert that is dropped intoa well.

The cross-sectional profile of the multi-chambered cell culture deviceand individual chambers, such as the internal chamber, can have anyshape, such as circular, triangle, square, rectangle, pentagon, hexagon,or other polygon as well as irregular.

The distinct chambers can have distinct cell cultures. The cells can beany type of cell ranging from immortalized cell lines to primary cellsto patient-derived cells. In some instances, a tissue culture from apatient can be included in a distinct chamber. The cell cultures caninclude a single type of cell or a combination of cells, such as 2, 3,or 4 different types in a co-culture. In some natural tissues, multiplecells may be present, and such tissues can be simulated with a similarcell type combination.

The multi-chamber cell culture device may be connected to a flow orpressure regulating system that can regulate the pressure across thedistinct chambers or within each distinct chamber. Pumps and valves canbe used to regulate the pressure. As such, operation of the device caninclude regulating the pressures inside each of the chambers such as theouter conduit. For example, a tissue such as liver or the kidney may beleaky, pressure control can be used to simulate such leakiness of thetissue. Also, some tissue like the brain and tumor can have very highpressures, which can be simulated with controlling the pumps and valves.The system can regulate the pressure in each of these distinct chambersas desired to mimic normal vs. diseased conditions.

The pressure can be selectively controlled by valves under the operationof a control system. The control system can include a memory devicehaving computer executable instructions for selectively controllingvalves of the device in order to regulate pressure. The valves can beprepared from the same or different materials as the body of the device,or they can be separate materials that are inserted into ports in thedevice at discrete locations. Each discrete chamber can have one or morevalves for pressure regulation, which may be located in the top wall.Two valves can be used as an inlet and an outlet, as illustrated. Thedevice may include a fluid pathway inlet and outlet with valves as wellas a pressure inlet valve and pressure outlet valve in the top wall.Fluid pathway valves can regulate fluid flow, while the pressure valvescan regulate pressure within the chambers.

The multi-chambered cell culture device can be used for any purposeinvolving cell culture. The multi-chambered cell culture device can beused in cell culture methods to simulate an organ. The methods caninclude testing one or more analytes for a presence or absence ofbiological response from the simulated organ. The biological responsecan be from the one or more analytes modulating a biological pathway,cell function, metabolic function, or toxicity. Any of the studiesdescribed herein or in the incorporated references can be performed withthe multi-chambered cell culture device. The one or more analytes can beprovided to the simulated organ in any possible manner, such as by beingintroduced into the outer chamber, one or more barrier layer chambers,or internal chamber.

The device can be used to test the effect of any substance on the cellsor simulated organ, and vice versa. The substance can be a biologicallyactive agent that can be any agent that is administered for a function,such as a biological function to improve or otherwise modulate abiological process, such as a biological pathway. However, the agent canbe active, such as to emit light, without being biologically active. Assuch, the biologically active agent can be a traditional pharmaceuticalor nutraceutical, and it can be any type of substance for testing ordiagnostics. The biologically active agent can be any agent that isadministered to a subject in order to elicit a biological response thatarises from the biological activity of the agent. The biologicalresponse obtained can be a measurable biological response or providesome change that can be analyzed and determined, such as by testing todetermine an amount of the biologically active agent to be administered.The biologically active agent can be a toxin or poison or otherdeleterious substance. Examples can include the biologically activeagent being a mineral, vitamin, pharmaceutical, nutraceutical, smallmolecule, macromolecule, organic molecule, polypeptide, protein, nucleicacid, polynucleotide, derivatives thereof, and combinations thereof. Thebiologically active agent can be for a human or animal subject. Humanand veterinary medicines can be evaluated and improved with the presentinvention. The substance can be an agricultural agent such asherbicides, pesticides, and/or fertilizers. The substance can be anenvironmental substance that is natural or manmade and found in theenvironment. The substance can be a particle. The substance can be aforeign cell not found in an organ, such as a cancer cell, bacteria,yeast, or the like, and even a virus. The test substance can be aparticle, such as a nanoparticle, liposome, microparticle, ormicrosphere or any other similar type of particle.

The test substance can even be a substance commonly used in apharmaceutical product or combination thereof to test for activity incertain simulated organs. The test substance can include the following:a film-forming agent; a filler; a plasticizer; a taste-masking agent; acoloring agent; a solubilizing agent; an effervescent agent; anantioxidant; an absorption enhancer; a disintegrating agent; a pHmodifying or buffer agent; a surfactant; a complexing agent; abioadhesive agent; a sheet adhesive; an identifying agent; ananti-counterfeiting agent; a tracking agent; transporter inhibitoragent; transporter inducer agent; emulsifying agent, self-emulsifyingsystem agents; crystallization inhibitor; crystallization promoter;supersaturation promoting agent; antimicrobial preservative; catalyst;chelating agent; particles; organoleptic agent; flavoring agent; scentagent; identifying device; and/or anti-counterfeiting device.

In one embodiment, cells can be analyzed in any of the chambers.However, in some assays, only the cells in the internal chamber will beassayed. For example, visual analysis, such as with a microscope can beused for analysis of the cells. In another example, the cells can beidentified using optical or electrical methods. For example, cellstaining markers specific for cell types can be used. In addition,electrical signals based detection can allow detection of morphologychanges (cell differentiation) and different types of cells.

In one embodiment, a cell culture device can include: a top wall; abottom wall; one or more perimeter walls coupled with and extendingbetween the top wall and bottom wall; and at least 3 distinct chambersbetween the top wall, bottom wall. The one or more perimeter walls caninclude: an internal chamber defined by at least one porous internalwall and having an internal chamber inlet and an internal chamberoutlet; one or more boundary layer chambers having at least an innerboundary layer chamber defined by the at least one porous internal walland at least one porous inner boundary layer wall, the at least oneporous internal wall having a plurality of pores fluidically couplingthe central internal chamber to the one or more boundary layer chamber;and an outer chamber defined by an outer porous boundary layer wall ofthe at least one porous boundary layer walls and the one or moreperimeter walls and having an outer conduit chamber inlet and an outerconduit chamber outlet, the outer porous boundary layer wall having aplurality of pores that fluidically couple the outer conduit chamberwith the one or more boundary layer chambers. In one aspect, wherein theat least three distinct chambers are nonlinear and/or idealized. In oneaspect, the internal chamber is central to and surrounded by the one ormore boundary layers. In one aspect, the internal chamber is concentricwith the one or more boundary layers. In one aspect, the internalchamber is central to and surrounded by the one or more boundary layersand outer conduit chamber. In one aspect, the internal chamber isconcentric with the one or more boundary layers and outer conduitchamber.

In one embodiment, the at least one porous internal wall includes poresdistributed from a first and to an opposite second end. The pores can beholes in the walls or gaps between posts or wall portions. In oneaspect, the at least one porous boundary layer wall includes poresdistributed from a first and to an opposite second end. In one aspect,all walls between the top wall, bottom wall, and one or more perimeterwalls are completely porous.

In one embodiment, the internal chamber inlet and outlet are at formedby internal chamber inlet walls and internal chamber outlet wallsextending between the top wall and bottom wall. In one aspect, the outerconduit chamber inlet and outlet are at formed by outer conduit chamberinlet walls and outer conduit chamber outlet walls extending between thetop wall and bottom wall. In one aspect, one or more of the boundarylayer chambers include a boundary layer chamber inlet and a boundarylayer chamber outlet. In one aspect, the boundary layer chamber inletand boundary layer chamber outlet are at formed by internal chamberinlet walls and internal chamber outlet walls extending between the topwall and bottom wall.

In one embodiment, one or more of the inlets and/or one of more of theoutlets are ports formed into one of the walls. In one aspect, the portsare formed into the top wall. In one aspect, one or more of the portsinclude a valve. In one aspect, one or more of the inlets and/or one ormore of the outlets include valves. In one aspect, one or more of theboundary layer chambers includes an inlet port having an inlet valveand/or an outlet port having an outlet valve.

In one embodiment, the internal chamber includes a first porous internalwall and opposite second porous internal wall with the internal chambertherebetween, and both the first porous internal wall and second porousinternal wall extending between the internal chamber inlet and outlet.

In one embodiment, the boundary layer chamber includes a first boundarylayer wall and opposite second porous boundary layer wall with theinternal chamber and boundary layer chamber therebetween. In one aspect,the boundary layer chamber encompasses the internal chamber. In oneaspect, both the first porous boundary layer wall and second porousboundary layer wall extending between the a boundary layer inlet andboundary layer outlet.

In one embodiment, at least one of the outer chamber, boundary layerchamber, or internal chamber, or inlet or outlet thereof, is configuredas an idealized microvascular network (IMN).

In one embodiment, at least one of the outer chamber, boundary layerchamber, or internal chamber, or inlet or outlet thereof is configuredas a synthetic microvascular network (SMN).

In one embodiment, the pores of the porous walls have a cross-sectionaldimension ranging from 5 nm to 500 microns. This can be the size of theposts. In one aspect, the porous walls have a width and/or height and/orthickness ranging between 5 microns and 500 microns.

In one embodiment, one or more of the outer chamber, boundary layerchamber, or internal chamber has a width ranging between 5 microns and500 microns.

In one embodiment, the bottom and side walls are integrated and the topwall is coupled to top ends of the side walls. In one aspect, the sidewalls, boundary layer walls, outer conduit walls, internal walls extendfrom and are integrated with the bottom wall.

In one embodiment, each wall separating adjacent chambers is porous. Inone aspect, the porous walls have pores located from the top wall to thebottom wall. In one aspect, the pores are contained within a wall. Inone aspect, the pores are gaps between adjacent wall segments.

In one embodiment, the cell culture device if operably coupled to a pumpsystem. In one aspect, the side walls, boundary layer walls, outerconduit walls, internal walls extend from and are integrated with thebottom wall. In one aspect, the inlets and/or outlets are operablycoupled to a pump system and analyte reservoir. In one aspect, valves inports of the top surface are coupled to a chamber pressure regulatingpump system.

In one embodiment, the device can include: a first cell culture in theouter chamber; a second cell culture in one or more of the boundarylayer conduits that is a different type from the first cell culture; anda third cell culture in the internal chamber that is a different typefrom at least one of the first cell culture and second cell culture. Inone aspect, the first, second, and third cell cultures are all differenttypes. In one aspect, the cell cultures include cells larger than thepores. In one aspect, the porous walls separating the one or morechambers include cells growing over the pores. In one aspect, the firstcell culture includes endothelial cells. In one aspect, the second cellculture include cells found in organ boundary layers. In one aspect, thesecond cell culture include cells found in organ boundary layersselected from brain, liver, heart, kidney, lung, stomach, intestines,pancreas, ovary, cervix, spleen, arteries, venules, capillaries and stemcells. In one aspect, the third cell culture include cells found inorgan tissue layers. In one aspect, the second cell culture includecells found in organ tissue boundary layers selected from epithelial,connective, muscle, bone and nervous tissue in addition to cellsrepresenting the germ layers orginating from stem cells. In one aspect,the combination of the first, second, and third cell cultures simulatesan organ from an endothelial surface, through organ boundary layers, andinternal cells. In one embodiment, an automated cell culture system 1250(See FIG. 12) can include: the cell culture device having one or moremulti-chambered constructs as described herein; and a computing system1260 (see FIG. 12) operably coupled to the cell culture device andhaving a memory device with computer executable instructions forcontrolling fluid flow of the chambers independently. The computingsystem 1260 can be configured to control any valve in any chamber forautomated cell culture methods for various assays. The computing system1260 and automated cell culture system 1250 can be utilized with any ofthe embodiments of the invention described herein.

In one embodiment, a cell culture device can include: a top wall; abottom wall; one or more perimeter walls coupled with and extendingbetween the top wall and bottom wall; and at least five distinctchambers between the top wall, bottom wall. The one or more perimeterwalls comprising: an internal chamber defined by at least one porousinternal wall and having an internal chamber inlet and an internalchamber outlet; three or more boundary layer chambers having at least aninner boundary layer chamber defined by the at least one porous internalwall and at least one porous inner boundary layer wall, the at least oneporous internal wall having a plurality of pores fluidically couplingthe central internal chamber to the one or more boundary layer chamber;and an outer chamber defined by an outer porous boundary layer wall ofthe at least one porous boundary layer walls and the one or moreperimeter walls and having an outer conduit chamber inlet and an outerconduit chamber outlet, the outer porous boundary layer wall having aplurality of pores that fluidically couple the outer conduit chamberwith the one or more boundary layer chambers. In one aspect, the atleast five distinct chambers are nonlinear SMN or idealized IMNconfigurations.

In one embodiment, one or more conduits include a substance. In oneaspect, one or more conduits include a gel. In one aspect, one or moreconduits include collagen. In one aspect, one or more conduits includelaminin. In one aspect, one or more conduits include matrigel. In oneaspect, one or more conduits are devoid of cells. In one aspect, one ormore conduits include a natural or synthetic biological matrix capableof cell culture. In one aspect, one or more conduits include abiological matrix configured for growing cells. In one aspect, one ormore conduits include a biological matrix and cells. In one aspect, someof the pores are filled with a gel. In one aspect, all of the pores in awall separating chambers are filled with a gel. In one aspect, some orall of the pores in a wall separating chambers are filled with matrigel.In one aspect, all of the pores in a wall separating chambers aresmaller than a cell. In one aspect, all of the pores in a wallseparating chambers are larger than a cell.

In one embodiment, the device can include one or more idealizedmicrovascular networks connected to one or more of the inlets or outletsof one of the chambers.

In one embodiment, the device can include one or more syntheticmicrovascular networks connected to one or more of the inlets or outletsof one of the chambers.

In one embodiment, a cell culture device can include: an onion-likechamber system having three or more of distinct chambers between a topwall, bottom wall, and one or more perimeter walls. The onion chambersystem can include: an internal chamber defined by at least one porousinternal wall and having an internal chamber inlet and an internalchamber outlet; three or more boundary layer chambers having at least aninner boundary layer chamber defined by the at least one porous internalwall and at least one porous inner boundary layer wall, the at least oneporous internal wall having a plurality of pores fluidically couplingthe central internal chamber to the one or more boundary layer chamber;and an outer chamber defined by an outer porous boundary layer wall ofthe at least one porous boundary layer walls and the one or moreperimeter walls and having an outer conduit chamber inlet and an outerconduit chamber outlet, the outer porous boundary layer wall having aplurality of pores that fluidically couple the outer conduit chamberwith the one or more boundary layer chambers. In one aspect, wherein theonion chamber is nonlinear or idealized. In one aspect, the device caninclude plurality of onion chamber systems coupled together. In oneaspect, the device can include a plurality of onion chamber systemsseparate from each other. In one aspect, the cells in conduits orchambers of the device can be identified using optical and/or electricalbased detection.

In one embodiment, the multi-chambered constructs can be used in a cellculture assay, which can include incubating calls in one or more of thechambers and testing an interaction of a substance with the cells of oneor more of the chambers. The substance can be any substance, and can bemolecule, protein, nucleic acid, cell, bacteria, virus, or other.

In one embodiment, a method of testing an analyte can include obtainingthe cell culture device having one or more multi-chambered constructs;introducing a test analyte into one of the outer chamber, boundary layerchamber, or internal chamber; incubating the test analyte with one ofthe first, second or third tissue cultures; and determining whether ornot the test analyte had an effect on the first, second, or third tissuecultures. In one aspect, the method can include simulating a pressureprofile across the outer chamber, boundary layer chamber, or internalchamber with the valves. In one aspect, the method can includefacilitating passage of the analyte into an adjacent chamber. In oneaspect, the method can include facilitating passage of the analyte intothrough an adjacent chamber and into a third chamber. In one aspect, themethod can include facilitating passage of a metabolite of the analyteinto an adjacent chamber. In one aspect, the method can includefacilitating passage of a metabolite of the analyte into an adjacentchamber into a third chamber. In one aspect, the method can includedetermining a change in one or more of the first, second, or third cellculture in response to the test analyte.

In one embodiment, and assay method can include: performing an assay tomeasure a parameter for one or more of the first, second, or third cellculture; and determining a difference in the parameter between one ofthe first, second, or third cell culture compared to another.

In one embodiment, an assay method can include: introducing a testreagent that interacts with the test analyte or metabolite thereof intoone of the first, second, or third cell culture; and detecting aninteraction between the test reagent and test analyte in one of thefirst, second, or third cell culture. In one aspect, the method caninclude tracking the test analyte passage between two of the outerchamber, boundary layer chamber, and internal chamber.

In one embodiment, an assay method can include introducing the testanalyte into the outer chamber; and determining an effect of the testanalyte or metabolite thereof on the third cell culture.

In one embodiment, an assay method can include simulating with thedevice an organ of a body.

In one embodiment, an assay method can include determining a diffusionparameter of the test analyte from the outer chamber, through theboundary layer chambers, and into the internal chamber.

In one embodiment, an assay method can include using optical and/orelectrical based detection to identify the cells in conduits or chambersof the device.

The present invention provides apparatus and methods that can be used tostudy fluid flow and particle adhesion in physiological vesselsincluding arterioles, capillaries, venules, and microvascular networkscomprising any combination of the three. The same apparatus and methodscan also be used to optimize drug delivery in the microvasculature. Manydrugs are available in microencapsulated, liposomal/lipisomal, and othermicro and nanoscale particle forms. The adherence and uptake of theseparticles in the microvasculature depends on specific and non-specificinteractions between the surfaces of drug delivery particles andendothelial cells that line the walls of the microvasculature. Adhesionalso depends on fluid dynamics parameters such as flow velocities andshear forces which, in turn, depend on vascular network geometries. Thepresent invention provides microfluidic chips comprising syntheticmicrovascular networks (SMNs) with flow channels that possess keygeometric and topological features that cause them to display the sametypes of fluid flow patterns and particle adhesion patterns as are foundin physiological microvascular networks. In addition, the SMNs requirequantities of reagents that are reduced by orders of magnitude comparedwith currently used techniques. Known microfabrication techniques alsoallow for development of plastic, disposable chips eliminating concernsof cross-contamination.

A “synthetic microvascular network” (SMN) is a man-made network thatcomprises a plurality of interconnected flow channels that formgeometrical features and have fluid flow properties found inphysiological microvascular networks. The flow channels (syntheticvessels) form intersecting networks and may be arranged end to end,analogous to an arteriole, capillary, venule sequence. Flow channels andthe SMNs they form possess one or more geometric characteristics ofphysiological microvascular selected from variable cross-sectionalshapes, variable cross-sectional areas, convolutions, turns, andanastomoses. A network consisting entirely of linear channels withconstant cross sectional areas, for example, is not a SMN because such anetwork does not possess the required physiological characteristics of aphysiological microvascular network. One or more flow channels of a SMNmay comprise walls made of a porous material such that fluid may movefrom the interior (lumen) of the flow channel into a space (e.g.,multi-chamber construct) external to the lumen in a manner similar tothe movement of fluid from the lumen of a physiological vessel into aninterstitial space, or vice versa.

An Idealized microvascular network (IMN) is a man-made networkcomprising interconnected flow channels that have certain fluid flowproperties found in physiological microvascular networks. The diametersof the channels range from 10-500 microns and comprise of anglestypically between 15 degrees and 135 degrees. Flow channels in amulti-chambered construct based on IMNs comprise porous walls (0.2-5microns) such that liquid may move between chamber, such as a mannersimilar to the movement of fluid from the lumen of a physiologicalvessel into an interstitial space or vice versa. As used herein, theterm “idealized” in association with a microfluidic network, junction,or bifurcation is used to describe a synthetic network, junction, orbifurcation consisting of straight microfluidic channels joined atacute, right, or obtuse angles.

As used herein, a microfluidic channel may have a rectangular, circular,semi-circular, irregular or a combination of cross-sectional shapes. Thedimensions of a channel are described, for example, by length, depth andwidth wherein the depth is measured perpendicular to the plane of amicrofluidic chip containing the channel and length and width aremeasured in directions lying in the plane of the microfluidic chipcontaining the channel. Channels having circular or semi-circularcross-sections may be described as having variable depth and widthrelative to channels having rectangular cross-sections or mayalternatively be described in terms of channel diameter. Maximum depthand width when used to describe a channel having a circular orsemi-circular cross-section are both equal to the maximum diameter ofthe channel. When used to describe a channel having a rectangularcross-section, the maximum width and depth refer to the constant widthand depth of a channel having a constant width and depth or to thehighest values for width and depth for channels having variable widthand depth.

In a non-limiting example, a microfluidic chip having one or moremulti-chamber constructs is constructed using techniques employed in thesemiconductor industry such as photolithography, wet chemical etching,thin film deposition and soft lithography using polymeric substrates,such as Polydimethylsiloxane (PDMS). Other materials that may be used inplace of PDMS include Poly(Styrene Butadiene Styrene) (SBS) andPoly(Styrene-Ethylene-Butadiene-Styrene) (SEBS) elastomers,Polyester-ether (PEE) thermoplast, and thermoset polyester (TPE), whichcan be used for replica molding fabrication techniques. Polyolefinplastomer (POP's) can be specifically used for submicron range channels.Glass or quartz with reactive wet/dry etching of the microchannels canalso be used. Thermoplastic materials such as polymethylmethacrylate(PMMA), polycarbonate (PC), cyclic olefin copolymer (COC), polystyrene(PS), poly vinyl chloride (PVC), and polyethylene terephthalate glycol(PETG) can be used with embossing techniques or injection molding. PS,PC, cellulose acetate, polyethylene terephthalate (PET), PMMA, PETG,PVC, PC, and polyimide can also be used with laser ablation techniques.In general, a microfluidic chip is formed with a number of microchannelsthat are connected to a variety of reservoirs containing fluidmaterials. The fluid materials are driven or displaced within thesemicrochannels throughout the chip using electrokinetic forces, pumpsand/or other driving mechanisms. Other manufacturing techniques can beused.

In one embodiment, the outer chamber can be devoid of an inlet and/oroutlet. In one embodiment, the one or more boundary layer chambers canbe devoid of an inlet and/or outlet. In one embodiment, the internalchamber can be devoid of an inlet and/or outlet. With the proviso thatthe multi-chamber cell culture device includes at least one inlet, andpreferably also includes at least one outlet.

From the foregoing, it will be appreciated that various embodiments ofthe present disclosure have been described herein for purposes ofillustration, and that various modifications may be made withoutdeparting from the scope and spirit of the present disclosure.Accordingly, the various embodiments disclosed herein are not intendedto be limiting, with the true scope and spirit being indicated by thefollowing claims.

All references recited herein are incorporated herein by specificreference in their entirety, including: U.S. Pat. No. 7,725,267; U.S.Pat. No. 8,175,814; U.S. 20100099136; U.S. 2009/0023608; U.S.2009/0203126; U.S. 20100112550; U.S. Ser. No. 13/332,400; U.S.20100227312; and U.S. 20110104658. The multi-chambered device can bedesigned, manufactured, and used in accordance with the principles andfor the study of diseases or disease states of the incorporatedreferences.

1. A cell culture device comprising: a top wall; a bottom wall; one ormore perimeter walls coupled with and extending between the top wall andbottom wall; and at least 3 distinct chambers between the top wall,bottom wall, and one or more perimeter walls comprising: an internalchamber defined by at least one porous internal wall and having aninternal chamber inlet and an internal chamber outlet; one or moreboundary layer chambers having at least an inner boundary layer chamberdefined by the at least one porous internal wall and at least one porousinner boundary layer wall, the at least one porous internal wall havinga plurality of pores fluidically coupling the central internal chamberto the one or more boundary layer chamber; and an outer chamber definedby an outer porous boundary layer wall of the at least one porousboundary layer walls and the one or more perimeter walls and having anouter chamber inlet and an outer chamber outlet, the outer porousboundary layer wall having a plurality of pores that fluidically couplethe outer chamber with the one or more boundary layer chambers, whereinthe at least 3 distinct chambers are nonlinear or idealized.
 2. The cellculture device of claim 1, wherein the internal chamber is central toand surrounded by the one or more boundary layers and outer chamber. 3.The cell culture device of claim 1, wherein the at least one porousinternal wall and/or at least one porous inner boundary layer wallincludes pores distributed from a first and to an opposite second end.4. (canceled)
 5. The cell culture device of claim 1, wherein one or moreof the boundary layer chambers include a boundary layer chamber inletand a boundary layer chamber outlet.
 6. (canceled)
 7. (canceled)
 8. Thecell culture device of claim 1, wherein at least one of the outerchamber, boundary layer chamber, or internal chamber, or inlet or outletthereof, is configured as an idealized microvascular network (IMN). 9.The cell culture device of claim 1, wherein at least one of the outerchamber, boundary layer chamber, or internal chamber, or inlet or outletthereof is configured as a synthetic microvascular network (SMN). 10.The cell culture device of claim 3, wherein: the pores of the porouswalls have a cross-sectional dimension ranging from 5 nm to 500 microns;or the porous walls have a width and/or height and/or thickness rangingbetween 5 microns and 500 microns. 11-15. (canceled)
 16. The cellculture device of claim 5, wherein the inlets and/or outlets areoperably coupled to a pump system and analyte reservoir.
 17. (canceled)18. The cell culture device of claim 1, comprising: a first cell culturein the outer chamber; a second cell culture in one or more of theboundary layer conduits that is a different type from the first cellculture; and a third cell culture in the internal chamber that is adifferent type from at least one of the first cell culture and secondcell culture. 19-21. (canceled)
 22. The cell culture device of claim 18,wherein the first cell culture includes endothelial cells, wherein thesecond cell culture includes cells found in organ boundary layers, andwherein the third cell culture includes cells found in organ tissuelayers. 23-26. (canceled)
 27. The cell culture device of claim 18,wherein the combination of the first, second, and third cell culturessimulates an organ from an endothelial surface, through organ boundarylayers, and internal organ tissue cells.
 28. An automated cell culturesystem, comprising: the cell culture device of claim 1; a computingsystem operably coupled to the cell culture device and having a memorydevice with computer executable instructions for controlling fluid flowof the chambers independently.
 29. A method of testing an analyte, themethod comprising: obtaining the cell culture device of claim 1;introducing a test analyte into one of the outer chamber, boundary layerchamber, or internal chamber; incubating the test analyte with one ofthe first, second or third tissue cultures; determining whether or notthe test analyte had an effect on the first, second, or third tissuecultures.
 30. A method of claim 29, comprising: simulating a pressureprofile across the outer chamber, boundary layer chamber, or internalchamber with the valves.
 31. A method of claim 29, comprising:facilitating passage of the analyte into an adjacent chamber;facilitating passage of the analyte through an adjacent chamber and intoa third chamber; facilitating passage of a metabolite of the analyteinto an adjacent chamber; or facilitating passage of a metabolite of theanalyte into an adjacent chamber into a third chamber. 32-34. (canceled)35. A method of claim 29, comprising: determining a change in one ormore of the first, second, or third cell culture in response to the testanalyte.
 36. A method of claim 29, comprising: performing an assay tomeasure a parameter for one or more of the first, second, or third cellculture; and determining a difference in the parameter between one ofthe first, second, or third cell culture compared to another.
 37. Themethod of claim 29, comprising: introducing a test reagent thatinteracts with the test analyte or metabolite thereof into one of thefirst, second, or third cell culture; and detecting an interactionbetween the test reagent and test analyte in one of the first, second,or third cell culture.
 38. The method of claim 29, comprising: trackingthe test analyte passage between two of the outer chamber, boundarylayer chamber, and internal chamber.
 39. The method of claim 29,comprising: introducing the test analyte into the outer chamber; anddetermining an effect of the test analyte or metabolite thereof on thethird cell culture.
 40. (canceled)
 41. The method of claim 29,comprising: determining a diffusion parameter of the test analyte fromthe outer chamber, through the boundary layer chambers, and into theinternal chamber.
 42. A method of claim 29, comprising using opticaland/or electrical based detection to identify the cells in conduits orchambers of the device.